Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular analyte or “antigen.” An antigen is a substance which is capable of inducing an immune response, i.e., antibody production, when introduced into an animal or human body. The region of a antigen that is recognized by an antibody, and to which the antibody binds, is referred to as an “epitope.”
The simplest immunoassay involves merely incubating an antibody that is capable of binding to a target molecule (i.e., the “analyte”) with a sample that is suspected to contain the analyte. The presence of the target molecule is determined by the presence, and is proportional to the concentration, of any immune complexes that form through the binding of the antibody and the analyte. In order to facilitate the separation of such immune complexes from the uncomplexed antibody, a solid phase is typically employed. In more sophisticated immunoassays such as particle-enhanced assays, the concentration of the target molecule is determined by binding the antibody to a support such as latex particles, and then incubating the support-bound antibody in the presence of the analyte-containing sample.
Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the presence of a labeled, second antibody (i.e., a “sandwich” immunoassay) that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the target in the sample. In an alternative assay, the sample is incubated with a known amount of labeled target and antibody binding site. The presence of any target molecules in the sample competes with the labeled target molecules for the antibody binding sites. Thus, the amount of labeled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecules in the sample. This is known as a competitive immunoassay.
The various immunoassay formats can be further divided into two main classes, depending upon whether the assay requires the separation of bound species from unbound species. Heterogeneous immunoassays require such purification, and hence entail a separation or isolation step. In contrast, homogeneous assays are designed such that the removal of bound species from unbound species is unnecessary. Because homogeneous assays lack a separation step, and are more easily automated, they are more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
In particle-enhanced immunoassays, an immune complex formation caused by a reaction between one or more particle-bound antibodies and the analyte results in particle aggregation. If the immune complex is large enough, it will become capable of scattering light, or of spontaneously precipitating. In such cases, agglutination, nephelometric, or turbidimetric detection methods may be employed. Nephelometric methods measure the light scattered by a suspension of particles or reflected toward a detector that is not in the direct path of light (Stemberg, J. C., Clin. Chem. 23: 1456-1464 (1977)). In contrast, turbidimetric methods measure the reduction of light transmitted through the suspension of particles or aggregates. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. Agglutination assays measure the precipitation of antibody-antigen complexes. Such assays can be extremely sensitive, and are amenable to automation. Because nephelometric and turbidimetric methods do not require the separation of the initially present antibody from the immune complexes formed in the assay, such assays are homogenous immunoassays.
Particle carriers typically used in such agglutination reactions are latex particles (e.g., particles of natural rubber or synthetic rubber latex, polystyrene latex, polyvinyltoluene latex). Polystyrene latex, being a synthetic product, has a longer shelf-life than non-latex carriers. In addition, latex securely binds proteins and other substances, and the antigenic properties of the bound proteins are substantially not impaired. Because of these desirable properties, latex particles have been employed as a raw material for a large variety of serological clinical test reagents.
It has been found, however, that latex particles sensitized with various antigens or antibodies often tend to undergo spontaneous aggregation during storage. In addition, even when a latex is of the type which does not undergo spontaneous aggregation during storage, it sometimes undergoes a non-specific aggregation upon admixture with body fluids such as serum. Non-specific aggregation interferes with the determination of an analyte's presence and/or concentration and can lead to an erroneous diagnosis. As a result, much time and effort has been expended in the search for the means of eliminating non-specific aggregation.
Currently known methods of reducing non-specific interferences in particle-enhanced assays include the following: addition of bovine serum albumin; massive dilution of the test sample up to at least 20-fold; addition of detergents such as are taught in U.S. Pat. No. 4,060,597; rigorous pre-treatment of the test sample including heat treatment for 30 minutes at 56° C. as described by Merz et at. (J. Clin. Micro., 5: 596 (1977)) enzymatic treatment with proteases reaction as described by Collet-Cassart et al. (Clin. Chem., 27: 1205, (1981)); treatment with reducing/oxidative reagents as described by Cambiaso et al. (J. Immuno. Meth. 28: 13, (1979)); and separation of components using ion exchange chromatography as described in U.S. Pat. No. 4,270,923. However, these methods do not work in all cases. In addition, these methods are time consuming and can carry with them the undesirable effect of drastically reducing the potential sensitivity and accuracy of the immunoassay as a result of the required manipulations.
Another approach to reducing non-specific particle aggregation has been the addition of specific chaotropic or chaotropic-like agents such as those described in U.S. Pat. No. 4,362,531 to de Steenwinkel et al. The described agents, however, include a wide range of dissimilar and unrelated compounds which are effective to varying degrees in relation to one another.
Ito et al. (U.S. Pat. No. 5,506,151) describe urea compounds useful as non-specific reaction suppressors. These urea compounds are hydrolysis products of carbodiimides, and these syntheses require the processing of large amounts of the hazardous and relatively expensive carbodiimide precursor.
For these reasons, the search for additives to aggregation reaction mixtures which reduce or eliminate the effects of non-specific interferences from physiological samples in such immunoassays continues.